Site-Selective Antibody Conjugation with Dibromopyrazines

In recent years, antibody conjugates have evolved as state-of-the-art options for diagnostic and therapeutic applications. During site-selective antibody conjugation, incomplete rebridging of antibody chains limits the homogeneity of conjugates and calls for the development of new rebridging agents. Herein, we report a dibromopyrazine derivative optimized to reach highly homogeneous conjugates rapidly and with high conversion on rebridging of trastuzumab, even providing a feasible route for antibody modification in acidic conditions. Furthermore, coupling a fluorescent dye and a cytotoxic drug resulted in effective antibody conjugates with excellent serum stability and in vitro selectivity, demonstrating the utility of the dibromopyrazine rebridging agent to produce on-demand future antibody conjugates for diagnostic or therapeutic applications.


General
Chemical reagents were purchased from Sigma Aldrich, Thermo Fisher and VWR International.Trastuzumab antibody was from Genentech Inc.The reactions were followed by analytical thin layer chromatography on silica gel 60 F254 and HPLC-MS chromatography with a Shimadzu LCMS-2020 device using a Reprospher 100 C18 (5 μm; 100 × 3 mm) column and positive-negative double ion source (DUIS) with a quadrupole MS analyzer in a range of 50-1000 m/z.High resolution mass spectra were recorded on a Waters Q-TOF Premier mass spectrometer in positive ESI ionization mode. 1 H and 13 C NMR spectra were recorded in CDCl 3 or DMSO-d 6 solution at room temperature, on a Varian Unity Inova 500 spectrometer (500 and 125 MHz for 1 H and 13 C/APT NMR spectra, respectively), and on a Varian Unity Inova 300 spectrometer (300, 75 MHz and 282 MHz for 1 H, APT NMR spectra, respectively), with the deuterium signal of the solvent as the lock and TMS as the internal standard.Chemical shifts (δ) and coupling constants (J) are given in ppm and Hz, respectively.All melting points were determined on a Jasco SRS OptiMelt apparatus (Stanford, CA, USA) and are uncorrected.
The antibody solutions were centrifuged with Eppendorf Centrifuge 5810 R.

SDS-PAGE and densitometry
Non-reducing glycine-SDS-PAGE at 10% acrylamide running were performed following standard lab procedures.A 4% stacking gel was used and a broad-range MW marker (4.6-300 kDa, ProSieve QuadColor Protein Marker, Lonza) was co-run to estimate protein weights.Samples (10 μL at 5 μM) were mixed with loading buffer (3 μL, composition for 6×SDS: 1 g SDS, 3 mL glycerol, 6 mL 0.5 M Tris buffer pH = 6.8, 2 mg Coomassie-blue R250 in 10 mL), heated at 65 °C for 5 minutes.Samples were subsequently loaded into the wells in a volume of 13 μL.All gels were run at constant 200 mA for 45 minutes.Gels were stained using a Coomassie stain (0,12 g Coomassie-blue G-250, 0,10 g Coomassie-blue R-250, 500 mL MeOH, 400 mL distilled water, 100 mL acetic acid), after washing it was rested at room temperature for 16 h in water-ethanol mixture.Then the gels were imaged using HP Laserjet 1132 MFP scanner at 600 dpi.Images were saved under default brightness, contrast, and gamma settings.Densitometry was performed using ImageJ.Background subtraction was achieved using the built-in plugin with a rolling ball radius of 30, sliding paraboloid, and smoothing.Brightness and contrast settings were auto adjusted within the software.

Photophysical measurements
Absorbance measurements were carried out on a Jasco V-700 spectrophotometer (standard cell quartz cuvette with 1 cm light path length, 1 nm bandwidth, 400 nm/min recording speed) operating at 21 °C.We measured the absorbance spectra from 220 nm to 750 nm.The UV/Vis absorbance measurements of trastuzumab conjugate were carried out on Thermo Scientific NanoDrop TM 1000 Spectrophotometer and SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices; San Jose, CA).Before the measurements buffer exchange was performed six times with Sartorius Vivaspin 500 10000 MWCO at 15000 g for 10 minutes each time.
The samples were buffer exchanged into ultrapure water and the concentration was set to 5 μM.
Before MS measurement, 30 μL of the trastuzumab samples were deglycosylated with 1 μL PNGase F (Glycerol Free) (New England Biolabs Gmbh, P0705), at 37 °C overnight.Flow rate was set to 0.5 ml/min.The column temperature was 60 °C and the injection volume was 10 µl.N 2 was used as the nebulizer gas (GS1), heater gas (GS2), and curtain gas with the optimum values set at 40, 45 and 40 (arbitrary units), respectively.Data were acquired in positive ESI mode in the mass range of m/z=500 to 5000, with 1 s accumulation time.The source temperature was 400°C and the spray voltage was set to 5000 V. Declustering potential value was set to 80 V. PeakView TM V.2.2 (version 2.2, Sciex, Redwood City, CA, USA) was used for deconvoluting the raw electrospray data to obtain the neutral molecular masses.

Results of HPLC-MS kinetic measurements
Table S2: GSH-reactivity and aqueous stability assay results of the covalent probes (14a-c)

Entry Compound k
N/D: not determined (due to high instability and/or hyperreactivity).The reactions faster than the minimal time window necessary to obtain LC-MS spectra are reported here with a half-life < 0.05 h, due to the minimal running time was 3 min.
of TCEP (0.02 μmol, 10 μL 2 mM TCEP stock solution in water) was added.The reaction mixture was incubated for 90 minutes at 37 °C under constant agitation (300 rpm).After this time, TCEP was removed using a ZebaSpin 7 kDa MWCO desalting column into pH=6 50 mM PBS buffer.To the reaction was added BUPY (0.2 µL, 10 Mm, 5 eq.) and was incubated for 90 minutes at 37 °C under constant agitation (300 rpm).After this time, the protein was purified from the small molecular reactants with VivaSpin 500 10 kDa MWCO membrane filter twice and ZebaSpin 7 kDa MWCO columns once.The conjugate was analyzed with UHPLC-MS, and non-reducing SDS-PAGE.The DOL was calculated from the MS spectrum.with 20 eq.BUPY and 5 µM mAb.g, Rebridging at pH=6 with 40 eq.BUPY and 5 µM mAb.
Cell lines and culture conditions MDA-MB-231 control human breast adenocarcinoma, SKOV-3 (HER2+) human ovarian adenocarcinoma cell lines were cultured in sterile culture flasks at 37 °C in a humidified atmosphere with 5% CO2 in an incubator.The cells were cultured in RPMI-1640 (Biosera, Cholet, France), supplemented with 10% FBS (Fetal Bovine Serum South American; Biosera, Cholet, France), Penicillin/Streptomycin Solution 100x (Biosera, Cholet, France).All work with the cell lines were performed in a laminar flow biosafety cabinet.Flow cytometrySamples were analysed using an Attune NxT flow cytometer (ThermoFisher Scientific).Trastuzumab fluorescence (BL1-H) was measured with 488 nm excitation and 530/30 nm emission.Analysis was performed using Attune NxT 3.1.2software.Preparing labelled cells with T-BUPY for FACS MDA-MB-231 control breast and SK-OV-3 (HER2+) ovarian carcinoma cells were harvested with acutase and centrifuged at 300 g for 5 min.One million cells per sample and three replicates per cell line were prepared.The cells were washed in phosphate buffered saline and the supernatant was washed off the cells after each washing step.After the washes, 100 µL of 3% bovine serum albumin (BSA) was added to the cells per tubes and incubated for 30 minutes to block non-specific bindings.After blocking, 100 µL of primary antibody (T-BUPY) (1:1000) was added to the tubes and incubated for 45 minutes at room temperature.The cells were washed three times with PBS, 100 µL of secondary antibody [Fluorescein (FITC) AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch Laboratories, 109-095-003, Lot.147561)] (1:200) was added and incubated in the dark for 45 minutes.After the incubation time, cells were washed three times with PBS, then resuspended in 300 µL PBS per sample.Preparing labelled cells with T-BUPY for FACS MDA-MB-231 (low HER2) breast and SKOV-3 (high HER2) ovarian carcinoma cells were harvested with acutase and centrifuged at 300 g for 5 min.One million cells per sample and three replicates per cell line were prepared.The cells were washed in phosphate buffered saline and the supernatant was washed off the cells after each washing step.After the washes, 100 µL of 3% bovine serum albumin (BSA) was added to the cells per tubes and incubated for 30 minutes to block non-specific bindings.After blocking, 100 µL of primary antibody (T-BUPY)(1:1000) was added to the tubes and incubated for 45 minutes at room temperature.The cells were washed three times with PBS, 100 µL of secondary antibody (Fluorescein (FITC) AffiniPure Goat Anti-Human IgG) (1:200) was added and incubated in the dark for 45 min.After the incubation time, cells were washed three times with PBS, then resuspended in 300 µL PBS per sample.ImmunocytochemistryGlass coverslips were sterilized in 90% ethanol, and after drying they were placed in 6 well cell culture plates (Starstedt, Nümbrech, Germany).100 µl fibronectin (Sigma, St. Louis, Missouri, USA) per well was added and incubated for 30 minutes in 37 °C termostate.Cells were suspended using trypsin-EDTA (Biosera, Cholet, France) and washed with PBS (Dulbecco's Phosphate Buffered Saline, Biosera, Cholet, France).Approximately 200.000 cells were placed in each well in 1ml of culture medium.After 24 hours of incubation at 37 °C, the cells were washed 3 times with PBS (Biosera, Cholet, France) and then fixed in 4% paraformaldehyde (Thermo Scientific, Waltham, Massachusetts, USA) for 10 minutes.After three PBS washes, we blocked the non-specific protein binding sites with 30 minutes of BSA (Sigma, St. Louis, Missouri, USA) incubation.The cells were incubated with the dye-conjugated primary antibody in 20 µg/mL concentration for 1 hour.After three PBS washes the samples were treated with Hoechst (Sigma, St. Louis, Missouri, USA) for nucleus staining and covered by ProLong Antifade Mountant (Thermo Scientific, Waltham, Massachusetts, USA).The samples were investigated with a Zeiss LSM 710 confocal microscope (Zeiss, Jena, Germany).MTT assayCell viability was determined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) which was obtained from Duchefa Biochemie (Haarlem, The Netherlands).After standard harvesting of the cells by trypsin-EDTA (Lonza) and phosphatebuffered saline (PBS, Lonza), 5 × 103 from MDA-MB-231 low HER2 breast (control) and 6×103 cells per well from SKOV-3, HER2 positive ovarium carcinoma cell lines were seeded in 5% serum containing growth medium to 96-well plates with flat bottom (Sarstedt), in a 100 μL volume per well, and incubated at 37 °C.After 24 h, cells were treated with various concentrations of compounds (50 μg/mL -0,2 μg/mL), dissolved in medium (0.5% final, Sigma Aldrich, St. Louis, MO, USA) and serum free medium (serum final 2.5%) and incubated for 72 h under standard conditions.The control wells were treated with serum free medium.Afterward, the MTT assay was performed, in order to determine cell viability, by adding 20 μL of MTT solution (5 mg/mL in PBS, 0.5 mg/mL final) to each well and after 2 h of incubation at 37 °C, the supernatant was removed.The precipitated purple formazan crystals were dissolved in 100 μL of a 1:1 solution of dimethyl sulfoxide (DMSO; Sigma Aldrich) -96% Ethanol (Molar Chemicals Kft., Hungary) and the absorbance was measured after 15 min.at λ = 570 nm by using The Spark microplate reader (TECAN, Morgen Hill, CA, USA).Average background absorbance (DMSO-Ethanol) was subtracted from absorbance values of control and treated wells, and cell viability was determined relative to untreated (control) wells where cell viability was arbitrarily set to 100%.Absorbance values of treated samples were normalized versus untreated control samples and interpolated by Dose-response curve non-linear 4parameter (variable slope) regression analysis with GraphPad Prism 6 software (GraphPad, La Jolla, San Diego, CA, USA) to generate sigmoidal dose-response curves from which the half maximal inhibitory concentration (IC50) values of the compounds were calculated.The experiments were done in triplicate and each experiment was repeated twice.

Figure
Figure S9.SDS-PAGE analysis of stability.A, Coomassie stained gel, B, fluorescence of gel recorded before staining.I. Protein marker, II.Native trastuzumab, III.Reduced trastuzumab, IV.Bovine serum, V. T-BUPY-TAMRA, The numbers from 0 to 7 refer to the days of T-BUPY-TAMRA incubation in bovine serum.